1. Technical Field of the Invention
This invention relates to the fields of hematology and immunology and to improvements in reagents used for analyzing blood cells. More specifically, the present invention relates to an improved diluent especially suited for enumeration and sizing of blood cells, determination of hemoglobin parameters and differentiation of leukocyte subpopulations in a single blood cell sample by means of suitable electronic instrumentation.
2. Discussion of the Prior Art
It is a common medical diagnostic procedure to analyze a blood sample of a patient in order to make certain classic determinations with respect to the blood sample. This procedure is an important tool for the physician. Important parameters are referred to as red blood cell count (RBC), hematocrit (HCT), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT) and mean platelet volume (MPV). These parameters are included in a complete blood count (CBC). Other important determinations are white blood cell count (WBC) and differentiation of the white blood cells into three or more subpopulations.
In general, blood cell analysis comprises diluting a blood sample with a fluid which functions as a diluent, analyzing one portion of the diluted blood sample for red blood cell and platelet parameters and contacting the other portion of the diluted blood sample with a lytic reagent to remove the erythrocytes and platelets to enable enumeration and differentiation of leukocytes. One of the earliest diluents reported was physiological saline. More advanced diluents for whole blood samples typically contained certain physiological salts, specific buffers, and preservative agents. In addition, the use of other reagents in blood cell analysis, including anticoagulants in the blood sample, detergents, monoclonal antibodies, dyes and stains results in unpredictable interactions among the biological sample and the chemical reagents, especially over varying operating temperatures.
Because of the sensitivity of erythrocytes, leukocytes, platelets and hemoglobin concentration to the chemical reagents, it has been very difficult to find a combination of chemical reagents that do not degrade or undesirably alter the characteristics of the cells to be analyzed and hemoglobin parameters, especially over varied operating temperature conditions. Frequently, a component is added, for example a preservative to maintain the stability of a reagent, but which might seriously and detrimentally affect physical parameters of the cells. Or a component is added to a diluent for a certain functional property, but that component will adversely affect other blood cell determinations. In one such example, the addition of a component to stabilize leukocytes has been found to adversely affect hemoglobin concentration determinations.
Because the cell's physical parameters are affected by the constituents of any diluent and by the method of analysis, whether automated, semiautomated or manual, prior art diluents have been formulated specifically for use with a specific test or specific instrument. These prior art diluents have not been universal or multipurpose in the sense that they could be used in various test instruments or with various test methods. For example, generally, a commercial diluent used for hematology analysis is different from one used for fluorescence flow cytometer analysis because of the problems of diluent fluorescence or compatibility of the diluent to fluorescence probes or monoclonal antibodies.
Many of the prior art diluents have one or more shortcomings depending upon the instrument, operating temperature, age of the sample, and the specific parameters of the sample which are to be determined. Blood diluents for use in automatic hematology instruments have been described in U.S. Pat. Nos. 3,962,125 to Armstrong; 4,185,964 to Lancaster; 4,213,876 to Crews et al.; 4,529,705 to Larsen; 4,617,275 to Matsuda et al.; 4,745,071 to Lapicola et al.; 4,485,175 to Ledis et al.; 4,346,018; 4,521,518 and 4,962,038 to Carter et al.; 4,968,629 to Lapicola; and 5,250,438 to Ryan. In addition, the following patents provide additional information concerning blood diluents and their use.
U.S. Pat. No. 4,255,385 to Stroupe et al. describes a method and reagent for determining hemoglobin in blood samples. The reference discloses a reagent for lysing and diluting a whole blood sample for cell analysis. The reagent contains potassium ferricyanide for the hemoglobin determination.
U.S. Pat. No. 4,506,018 to North describes a blood diluent for avoiding blood cell volume changes by balancing the effects of a preservative and a surfactant used in the diluent.
U.S. Pat. No. 4,656,139 to Matsuda et al. describes a method of preparing cells for blood analysis comprising the steps of diluting a blood sample with a diluent comprising boric acid buffer solution, ethylenediaminetetraacetic acid (EDTA) and (2-pyridylthio-1-oxide) sodium. The (2-pyridylthio-1-oxide) sodium is employed to enable a hemoglobin determination.
U.S. Pat. No. 5,008,202 to Edmondson et al. describes a blood diluent and method for diluting blood for use in blood cell analysis. The blood diluent generally comprises an organic buffer, a cell stabilizing agent, an inorganic salt, a solvent and EDTA, wherein EDTA serves as an antimicrobial agent, or a mixture of EDTA and sodium fluoride wherein together they serve as an antimicrobial agent.
U.S. Pat. No. 5,227,304 to Wong describes a complicated reagent system for enumeration and sizing of blood cells in a Cell Dyn.RTM. brand blood analyzer. The reagent system comprises an isotonic diluent and detergent, wherein the diluent comprises an imidazole organic buffer, antimicrobial agent, inorganic salts and pH adjusting agent. In the specification, Wong teaches EDTA as a chelator and to enhance the antimicrobial character of the diluent, wherein the antimicrobial agents are sodium omadine 2-pyridinethiol-1-oxide! and Triadene 3 hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine! or Triadene 10 a mixture of 60% Triadene 3 and 6% sodium omadine!. (Sodium Omadine is a registered trademark of the Olin Corporation.)
U.S. Pat. No. 5,242,832 to Sakata describes a lytic reagent containing imidazole and imidazole derivatives as a hemoglobin stabilizer.
PCT Patent Application WO 95/24651 to Kim et al. describes a lytic reagent for use in total hemoglobin measurements containing lauryl dimethylamine oxide as a lytic agent and imidazole and its derivatives as a hemoglobin stabilizer. The lytic reagent is used to lyse erythrocytes for hemoglobin determinations without using cyanide. However, this reagent system is not used for any other blood cell analysis such as WBC or leukocyte differentiation.
Notwithstanding the prior art diluents, there still exists a need for a multipurpose diluent which minimizes the need for several diluents and which can be used in a varied temperature environment. A multipurpose diluent avoids the costs, inconvenience and potential confusion caused by stocking a variety of different diluents within a given clinical laboratory to accommodate the requirements of a specific hematology analyzer or flow cytometer analyzer and specific analysis performed on each such analyzer.